The identification and characterization of proteins and peptides has become a significant part of modern biology, and mass spectrometry has become one of the most important techniques used for the analysis of peptides and proteins. Recently, a novel means of peptide ion dissociation, referred to as electron transfer dissociation (ETD) was described (Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proceedings of the National Academy of Sciences of the United States of America 2004, 101, 9528-9533; Coon, J. J.; Syka, J. E. P.; Schwartz, J. C.; Shabanowitz, J.; Hunt, D. F. International Journal of Mass Spectrometry 2004, in press 2004). In ETD anions are reacted with multiply protonated peptide/protein cations in a linear ion trap mass spectrometer. The result is the transfer of an electron from the anion to the peptide. Following electron transfer, the peptide dissociates through the same pathways accessed in electron capture dissociation (ECD) (Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. Journal of the American Chemical Society 1998, 120, 3265-3266).
ETD is fast and efficient, allowing its direct implementation with chromatography for peptide sequence analysis. Furthermore, ETD dissociates intact proteins with similar efficiency as the smaller peptides described in earlier work (Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proceedings of the National Academy of Sciences of the United States of America 2004, 101, 9528-9533). FIG. 2 displays the products obtained following a 15 ms reaction of the ETD-inducing anion, fluoranthene, with the +10 cation of residues 1-52 of histone H4 (SGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARR GGVKRISGLIYE; SEQ ID NO: 2). Observed are hundreds of c and z-type fragment ions, many of which are multiply charged. In fact, most of these product ions are highly charged. To fully separate the multiple isotopic peaks associated with the fragment species requires m/z resolving power beyond that available from linear ion trap mass spectrometers. Direct ETD dissociation of large peptide/protein cations, including for example, residues 1-52 of histone H4, typically generate product ion spectra that are too complicated to yield sequence information. Namely, this limitation is due to the presence of dozens or hundreds of highly charged c and z-type fragments all clustered within the ˜300-1000 m/z range. Thus, without introduction of a second mass analyzer (hybridization) capable of resolving this complicated mixture of fragment ions, the practical applicability of direct ETD fragmentation of large peptide/protein cation is somewhat limited, especially for sequencing a priori unknown proteins.
In addition to the recently discovered ETD reaction, another type of ion/ion reaction was described several years ago by McLuckey and co-workers (Stephenson, J. L.; McLuckey, S. A. Analytical Chemistry 1996, 68, 4026-4032; McLuckey, S. A.; Stephenson, J. L. Mass Spectrometry Reviews 1998, 17, 369-407). In that reaction, multiply charged peptide or protein cations are reacted with an anion that removes protons from the protein cation (proton transfer reactions, PTR). By removing protons from the highly charged protein cations the net charge of the protein is reduced. In this fashion, the charge state of the protein can be determined. McLuckey et al. have also used the PTR reaction to reduce the charge of protein fragment ions derived from collision-activated dissociation (CAD) (Reid, G. E.; McLuckey, S. A. Journal of Mass Spectrometry 2002, 37, 663-675; Reid, G. E.; Shang, H.; Hogan, J. M.; Lee, G. U.; McLuckey, S. A. Journal of the American Chemical Society 2002, 124, 7353-7362; Amunugama, R.; Hogan, J. M.; Newton, K. A.; McLuckey, S. A. Analytical Chemistry 2004, 76, 720-727).
Accordingly, highly charged b and y-type fragment ions derived from collision-activated dissociation can be reduced to singly charged species for easier interpretation. However, the use of CAD for the production of product ions suffers from several disadvantages including the following:
a) Peptides with post-translational modifications (i.e., phosphorylation and glycosylation, etc) often fragment by loss of the modification rather by cleavage of the peptide backbone. Only a relatively small percentage about (20%-30%) of these types of peptide ion precursors produce interpretable/searchable product ion spectra. This is somewhat lessened (less tendency for modification loss) as the number of amino acids in the peptide increases.
b) Peptides that contain multiple basic amino acid residues (Lys, Arg, and His) and thus carry more than two charges, also fail to fragment randomly along the peptide backbone and thus afford incomplete sequence information when analyzed by the above technology (CAD).
c) Peptides that contain more than 40 amino acids also fail to fragment randomly along the peptide backbone. These also afford incomplete sequence information.
Therefore, CAD fragmentation for protein cations usually does not provide adequate information regarding post-translational modification of the proteins and does not routinely cleave each peptide bond in the protein/large peptide. Typically only a few b and y-type cleavages are observed for large polypeptide species (e.g., greater than 40 amino acids) and the process is highly dependent upon the initial charge state of the protein (Hogan, J. M.; McLuckey, S. A. Journal of Mass Spectrometry 2003, 38, 245-256). Because of the random, non-predictable nature of CAD cleavage this type of experiment has not become a routine tool for whole protein sequence identification.
There is a long felt need in the art for the development of new methods for rapid sequence analysis of intact proteins or peptides or large protein degradation products. The present invention satisfies this need.